1 Highly efficient one-pot bioethanol production from corn stalk with

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2 biocompatible ionic liquids
3 Qingqing Zhua,b, Xingmei Lua,b, Die Gaoa, Dongxia Yana,c, Jing Tanga, Xiujie Chenga,
4 Ibrahim EI Tantawy EI Sayedd, Nour Sh. El-Gendye,f, Jiayu Xin*a,b and Suojiang Zhang*a,g

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5 aBeijing Key Laboratory of Ionic Liquids Clean Process, CAS Key Laboratory of Green
6 Process and Engineering, State Key Laboratory of Multiphase Complex Systems, Innovation
7 Academy for Green Manufacture, Institute of Process Engineering, Chinese Academy of
8 Sciences, Beijing 100190, China.

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9 bSino Danish College, University of Chinese Academy of Sciences, Beijing 100049, China.

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10 cLangfang Technological Centre of Green Industry, Langfang 065006, China.

11 dChemistry Department, Faculty of Science, Menoufia University, Shebin El-Kom, Egypt.

12 eEgyptian Petroleum Research Institute (EPRI), Nasr City, Cairo, Egypt, PO 11727

13 fCenter of Excellence, October University for Modern Sciences and Arts (MSA), 6th of October
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14 City, Giza, Egypt, PO 12566.
15 gHenan University, Zhengzhou 450001, China.

16 *E-mail: jyxin@ipe.ac.cn (J. Xin), sjzhang@ipe.ac.cn (S. Zhang); Tel./fax: +86-010-82627080

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17 Abstract
18 The transformation of lignocellulose into biofuel is of great significance as the world’s
19 nonrenewable resources run down. However, the efficiency of ionic liquids (ILs) and the cost
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20 caused by washing and separation of ILs are urgent problems that need to be solved. In this work,
21 one-pot bioethanol production with biocompatible ionic liquids was proposed. We found that the
22 crystallinity (from 0.68 to 0.43) and thermal stability (initial decomposition temperature from
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23 280°C to 275°C and melting point decreased) of corn stalk after being pretreated by [Ch][Gly]
24 trend to become easier to depolymerization characterized by XRD, TGA, and DSC. Under a one-
25 pot process, [Ch] [Gly] pretreatment efficiency could achieve a 9-fold sugar yield, i.e., 324.7 mg
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26 glucose/PU [Ch] [Gly] and 131.8 mg xylose/PU [Ch] [Gly] (1PU IL=10 g, 3 wt% [Ch] [Gly]
27 aqueous). The conditions for the whole process were optimized (pretreatment: 140°C, 2h, 10 wt%
28 biomass loading, 3 wt% [Ch][Gly]; saccharification: 50°C, 120 h, 25 mL water, 1 MPa CO2
29 pressure, 40 mg enzymes/g biomass; fermentation: 30 wt% yeast loading ) and the glucose,
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30 xylose, and bioethanol yield could be up to 80%, 79%, and 84%, respectively. Besides, we found
31 that the accessibility and lignin distribution were important to determining enzymatic digestibility
32 by LSCM and BET. Increasing surface area and pore size could enhance accessibility for enzymes
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33 to attack cellulose and hemicellulose.

34 Keywords: Biomass, Corn stalk, Bioethanol, Biocompatiable ionic liquids, One-pot

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35 1 Introduction

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36 Bioethanol is a potential biofuel for resolving the energy crisis, and bioethanol yields from both starch
37 and lignocellulose have increased rapidly as a result of widespread attention and investment [1].

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38 Lignocellulose, including dedicated energy crops, agricultural wastes, organic municipal solid waste,
39 and forestry wastes, is the most abundant biopolymer available on earth as waste biomass, and
40 research on it has a long tradition [2]. The recalcitrance of lignocellulose is a serious obstacle to
41 fermentable sugars by enzymolysis in crops because of the strong rigidity of lignin. The dilute sulfuric
42 acid pretreatment method has been widely used to weaken the recalcitrance of lignin to obtain

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43 fermentable sugars [3]. A well-known problem with this method is that the by-products such as

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44 furfural, 5-hydroxymethylfurfural (HMF), formic acid, acetic acid, levulinic acid, and phenolic
45 compounds would hinder microbial growth [4]. There are many alternative methods available for
46 biomass pretreatment, such as steam explosion, hot water, organic acids, ammonia-based and ionic
47 liquids [5]. A number of studies have shown that using ILs to pretreat lignocellulose is one of the
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48 most promising approaches due to the modifiable ions, better thermal stability, less volatility, and
49 higher biomass solubility [6]. Within 3 hours of exposure at 120°C, the 1-n-ethyl-3-
50 methylimidazolium acetate ionic liquid ([Emim][OAc]) was observed to quickly swell the secondary
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51 cell walls and completely solubilize the plant cell walls. The pretreated material was easy to hydrolyze
52 into glucose using the commercial cellulase cocktail, indicating that this method was Another IL 1-
53 ethyl-3-methylimidazolium acetate ([C2mim] [OAc]) has been studied to be effective at
54 pretreating biomass and has been demonstrated to scale up to larger volumes [8]. The inhibition
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55 of enzyme activity [9] and microbial toxicity [10] are the main hurdles for conventional ionic
56 liquids to achieve a cost-effective IL-based biomass conversion technology. Choline and amino
57 acid-based ionic liquids were reported as a good choice for biocompatible no-detox pretreatment due
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58 to its low toxicity [11]. Furthermore, it has been demonstrated that choline glycine ([Ch][Gly]) has
59 high lignocellulose pretreatment efficiency [12] and a variety of excellent physicochemical properties
60 (low viscosity: 121 mPa s, 25°C; high lignin solubility: 220 mg/g, 90°C). Besides, a series of low-
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61 toxicity protic ionic liquids (PILs) were synthesized by some simple amino bases (e.g., cholinium
62 hydroxide, diethanolamine, ethanolamine, and triethanolamine) and acids (e.g., acetic acid, sulfuric
63 acid, phosphoric acid, hydrochloric acid, and formic acid) [13]. Among these PILs, ethanolmine
64 acetate ([EOA][OAc]) and cholinium acetate ([Ch][OAc]) could get better sugar yields and achieve
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65 one-pot bioethanol production without removing lignin [14]. In another study, the efficiency of ionic
66 liquid pretreatments was enhanced after adding moderate acetic acid [15]. Therefore, [Ch][Gly],
67 [EOA][OAc] and [Ch][OAc] were selected to explore their application in one-pot bioethanol
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68 production.

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 69 The one-pot bioethanol process needs no separation and washing, thus reducing the cost of water and

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70 wastewater treatment compared with conventional processes. To achieve one-pot bioethanol
71 production, a pH adjustment stage is necessary before saccharification and fermentation because
72 enzymes and microorganisms live in a weakly acidic environment (pH = 4-6) in the acid buffer
73 solution (e.g. acetic acid-odium acetate, citric acid-odium citrate, monopotassium phosphate), while

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74 the bionic liquids are always strongly alkaline (pH = 10-12). Conventional buffer solutions would
75 exchange with the bionic liquids to destroy their structures. Some ionic liquids can reduce pH by
76 trapping large amounts of CO2 to form the corresponding carbonates under mild conditions [16]. The
77 process could be reversible when the temperature is high (70-100°C) or nitrogen is injected into the

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78 process, as previously reported for other ILs [17]. In the study of Sun et al., the [Ch][Lys] ionic liquid
79 was recycled succcessfully, and the ethanol yield of recycled [Ch][Lys] was about 80% by using CO2

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80 to adjust the pH value [18]. Besides, catching CO2 with ionic liquids could limit and alleviate the
81 global warming trend [19]. With this pH adjustment, it is possible to produce ethanol in a single pot.

82 This study achieved one-pot bioethanol production for the first time by destroying the structure of
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83 corn stalk using low-concentration bionic liquids ([Ch] [Gly], [EOA] [OAc], and [Ch] [OAc]. The
84 structure of the corn stalk before and after pretreatment by bionic liquids was analyzed by various
85 characterization methods (compositional analysis, XRD, TGA, and DSC). To further understand how
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86 to improve enzymolysis efficiency by a one-pot process, the SEM and LSCM analyses were used to
87 observe the morphology and lignin distribution of corn stalk at different stages (pretreatment and
88 enzymolysis). And this method was applied to different types of biomass to verify its universality.
89 Besides, the effect of conditions (pretreatment, enzymolysis, and fermentation) was explored. This
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90 study provided a potential strategy for simplified detoxification in the field of lignocellulosic
91 biorefinery.
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93 2 Experimental
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94 2.1 Materials
95 Corn stalk (CS), cotton stalk and reed stem were obtained from fields in Suzhou, Anhui Province in
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96 China. They were stored in glass containers after washing, drying, milling, and sieving (<0.125mm).
97 Glycine (Sinopharm Group Chemical Reagent Co., Ltd. (China)). Choline hydroxide (40% aqueous
98 solution) (Shandong Shouguang Luke Chemical Co., Ltd. (China)). Ethanol (Sigma-Aldrich) was
99 used without further purification. Glucose, xylose and arabinose (Shanghai Aladdin Biochemical
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100 Technology Co., Ltd. (China)). Cellulase and hemicellulase (Shanghai Macklin Biochemical Co., Ltd.
101 (China)).

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102 2.2 Experimental process

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103 Synthesis and purification of bionic liquids: Aqueous solutions of glycine and choline hydroxide
104 (molar ratio = 1:1.05) were mixed and stirred for 48 hours at 25°C and 800 rpm.The aqueous choline
105 glycine solution was concentrated with a rotary evaporator (RE 2000, Shanghai Yarong Biochemical

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106 Instrument Factory, China, vacuum 0.1 MPa, 70°C) and then washed three times with
107 acetonitrile/methanol (9:1, v/v). Subsequently, the organic solvent was removed by rotary
108 evaporation at 40°C and dried at 70°C (vacuum 0.1 MPa) for 2 days, with phosphorus pentoxide as
109 the desiccant. When the water content was below 10%, the ionic liquid was analyzed for water content
110 using a water partition tester. The synthesis methods of ethanolamine acetate and choline acetate ionic

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111 liquids are the same as for choline glycine ionic liquid.

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112 Compositional analysis of biomass: Before the experiments, the compositions of corn stalk, cotton
113 stalk, and reed stem were determined, which were analyzed by National Renewable Energy
114 Laboratory (NREL) acidolysis protocols (LAP) [20]. 0.15-0.16 g of corn stalk and 1.5 mL of 72 wt%
115 H2SO4 were incubated at 30℃ for 1 h, with stirring with a glass stick to avoid powder floating. The
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116 solution was diluted to 3.4 wt% H2SO4 with 42 mL of deionized water and autoclaved for 45 min at
117 120°C. The hydrolysate is separated by filtration. The quality of solid residue as Euqation (1) was
118 used to calculate the cellulose, hemicellulose, and lignin content. The pH value of supernatants was
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119 adjusted to 2. Glucose, xylose, and ethanol concentrations in the supernatants were determined using
120 a High Performance Liquid Chromatography (HPLC). Set the parallel experiment to take the average
121 value.

𝑀𝐹𝑢𝑛𝑛𝑒𝑙 ‒ 𝑀𝐹𝑢𝑛𝑛𝑒𝑙 + 𝑟𝑒𝑠𝑖𝑑𝑢𝑒

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122 Lignin (%） = × 100%#(1)

𝑀𝑠𝑎𝑚𝑝𝑙𝑒

𝐶𝑔𝑙𝑢 × 87 × 10 ‒ 3 × 0.9
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123 Cellulose (%) = × 100%#(2)

𝑀𝑠𝑎𝑚𝑝𝑙𝑒

(𝐶𝑎𝑟𝑎 + 𝐶𝑥𝑦𝑙) × 87 × 10 ‒ 3 × 0.88

124 Hemicellulose (%) = × 100%#(3)
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𝑀𝑠𝑎𝑚𝑝𝑙𝑒

125 where 𝑀𝑠𝑎𝑚𝑝𝑙𝑒 is the weight of the sample. 𝑀𝐹𝑢𝑛𝑛𝑒𝑙 is the funnel weight. 𝑀𝐹𝑢𝑛𝑛𝑒𝑙 + 𝑟𝑒𝑠𝑖𝑑𝑢𝑒 is the
126 funnel and residue weight after drying. 𝐶𝑔𝑙𝑢 , 𝐶𝑎𝑟𝑎and 𝐶𝑥𝑙𝑦 are glucose, arabinose, and xylose
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127 concentrations, respectively, which are evluated by HPLC.

128 Pretreatment of corn stalk with ILs: For example, CS (0.25 g) was blended with 5 g [Ch][Gly]
129 (3wt%) in a capped glass pressure tube and pretreated at 140°C for 2 h. The pretreated corn stalk was
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130 collected by centrifugation at 6000 rpm for 10 min and dried at 80°C in a drying oven for further
131 analysis. The composition analysis of corn stalk after pretreatment has been introduced in part 3.2.

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132 Adopting a control single variable method, [Ch][Gly] concentration, reaction time, temperature, and

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133 biomass loading were optimized.

134 Using CO2 to adjust pH: The CO2 absorption experiments were carried out at 25°C in a 50 mL
135 reactor (316 L stainless steel) equipped with a pressure gauge and CO2 cylinder (>99.99% CO2 purity).

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136 Firstly, the pH of different [Ch][Gly] concentrations (0 wt%, 1 wt%, 3 wt%, 5 wt%, and 10 wt%)
137 under 1 MPa CO2 was studied, and the corresponding pH value of the mixture was quickly measured
138 by pH Meter. Then, the mixed liquor after pretreatment was diluted with deionized water and poured
139 into the stainless-steel reactor. The sealed reactor was stirred at 50°C and the pressure was held
140 constant by a backpressure valve. To analyze the interaction between CO2 with the side amine and

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141 terminal amine groups of [Ch][Gly] in H2O, 1H NMR and FT-IR analyses for [Ch][Gly] before and

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142 after CO2 absorption were conducted.

143 One-pot pretreatment and saccharification process: The corn stalk (1.0 g) was mixed with 10.0 g
144 [Ch][Gly] (3 wt%) in a 15 mL capped glass pressure tube and the pretreatment conditins were 140℃
145 for 2 h. The pretreated slurry was diluted with water to obtain a 0.9 wt% final IL concentration. The
146
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saccharification conditions (40 mg enzyme cocktail loading, cellulase: hemicellulose=3:1, w/w) were
147 50°C for 120 h with constant agitation in the oil bath. In addition, the saccharification conditions (e.g.
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148 water amount, enzymolysis time, enzyme loading, temperature, and CO2 pressure) were optimized.

149 (C6H10O5)n + nH2O→nC6H10O6#(4)

150 (C5H8O4)m + mH2O→mC5H10O5#(5)

151 The glucose yield and xylose yield were calculated.

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𝐶𝑔𝑙𝑢 × 𝑚ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑎𝑡𝑒 × M𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑢𝑛𝑖𝑡

152 Glucose yield (%) = × 100% #(6)
𝑚𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 × M𝑔𝑙𝑢
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𝐶𝑥𝑦𝑙 × 𝑚ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑎𝑡𝑒 × Mℎ𝑒𝑚𝑖𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑢𝑛𝑖𝑡

153 Xylose yield (%) = × 100% #(7)
𝑚ℎ𝑒𝑚𝑖𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 × M𝑥𝑦𝑙
154 Where, 𝐶𝑔𝑙𝑢 and 𝐶𝑥𝑦𝑙 are glucose concentration, which are calculated by HPLC. 𝑚ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑎𝑡𝑒 is the
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155 weight of hydrolysate. M𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑢𝑛𝑖𝑡 and Mℎ𝑒𝑚𝑖𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑢𝑛𝑖𝑡 are the molar mass of one cellulose unit.
156 𝑚𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 and 𝑚ℎ𝑒𝑚𝑖𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 are the weight of cellulsoe in rich-cellulose. M𝑔𝑙𝑢 and M𝑥𝑦𝑙 are the molar
157 mass of glucose.
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158 One-pot pretreatment and saccharification fermentation process: The corn stalk (1.0 g) was
159 mixed with 10.0 g [Ch][Gly] (3 wt%) in a 15 mL capped glass pressure tube, and the pretreatment
160 conditions were 140℃ for 2 h. The pretreated slurry was diluted with water to obtain a 0.9 wt% final
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161 IL concentration. The saccharification conditions (40 mg enzyme cocktail loading, cellulase:
162 hemicellulose=3:1, w/w) were 50°C for 120 h with constant agitation in the oil bath. After

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163 enzymolysis, the yeast (Saccharomyces cerevisiae) was added into the hydrolysate at 33% yeast

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164 loading at 37°C for 48 h. The initial parameters for fermentation (e.g., yeast loading and fermentation
165 time) were optimized.

166 C6H10O6→2C2H5OH + CO2#(8)

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167 C5H10O5→ C2H5OH + CO #(9)
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168 Because the yeast could only ferment xylose, the theoretical ethanol mass was calculated when

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169 cellulose was completely transfromed. The ethanol yield equation was:

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𝐶𝑒𝑡ℎ𝑎𝑛𝑜𝑙 × 𝑚ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑎𝑡𝑒 × M𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑢𝑛𝑖𝑡
170 Ethanol yield (%) = × 100% #(10)
2 × 𝑚𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 × M𝑒𝑡ℎ𝑎𝑛𝑜𝑙
171 Where, 𝐶𝑒𝑡ℎ𝑎𝑛𝑜𝑙 is ethanol concentration, which are calculated by HPLC. 𝑚ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑎𝑡𝑒 is the weight
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172 of hydrolysate. M𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑢𝑛𝑖𝑡 us the molar mass of one cellulose unit. 𝑚𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 is the weight of
173 cellulsoe in rich-cellulose. M𝑒𝑡ℎ𝑎𝑛𝑜𝑙 is the molar mass of ethanol.
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174 2.3 Analysis and characterization methods
175 HPLC (High Performance Liquid Chromatography): A High Performance Liquid
176 Chromatography (Waters e2695, USA) equipped with a Bio-Rad Aminex® HPX-87H column and a
177 differential refractive index detector was used. An aqueous solution of H2SO4 (4 mM) was used as
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178 the mobile phase (0.4 mL min-1, column temperature 50°C). The injection volume was 20 µL with a
179 run time of 35 min. The appearance times of glucose, xylose, arabinose, and ethanol peaks are 13
180 min, 14 min, 15 min, and 33 min, respectively.
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181 XRD (X-ray diffraction): A multifunctional X-ray diffractometer (X-Pert PRO MPD) and MDI
182 Jade 6 Software were used to measure the crystallinity of corn stalks before and after they were treated.
183 The patterns were collected in the 2θ range from 5° to 90°. Scanning speed was 4° min-1. The recorded
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184 diffraction peaks were compared with the standard peaks to determine their attribution.

185 TGA (Thermogravimetric analyser) and DSC (differential scanning calorimetry ): TGA and
186 DSC analyses were performed on the German STA449F3 synchronous thermal analyser to investigate
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187 the thermal behaviour of corn stalks before and after pretreatment with BILs. 5 mg of sample was
188 heated at a rate of 10 K min−1 from 25°C to 600°C and a flow rate of 50 mL min−1 in a weighting
189 alumina pan under a nitrogen atmosphere. Furthermore, the melting point and heat of fusion were
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190 analyzed by TA universal analysis software. And the 5% weight loss was set as the initial
191 decomposition temperature.

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192 SEM (Scanning electron microscope): A cold field emission scanning electron microscope (JEOL

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193 JSM-6700F) was used to characterize the morphology of the samples. The corn stalks before and after
194 pretreatment were laid flat on conductive adhesive and then sprayed with gold. After the samples
195 were sprayed with gold, they were put under a 5 kV electron microscope to observe the morphology
196 and take pictures.

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197 BET (Brunauer-Emmett-Teller technique): The corn stalks (100 mg) before and after pretreatment
198 were dried at 200 ℃ under vacuum conditions. The surface area and pore size distribution were
199 analysed by BET (Autosorb iQ, USA) using chemisorption temperature reduction (TPR). The
200 samples after analysis could be recycled.

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201 NMR (Nuclear Magnetic Resonance): The samples were characterized at room temperature using

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202 a liquid superconducting NMR spectrometer BRUKER AVANCE-III 700 M (Bruker, Germany).

203 LSCM (Laser Scanning Confocal Microscopy): Corn stalk particles were imaged with Laser
204 Scanning Confocal Microscopy (Leica TCS SP 5, Germany). Lignin is a complex heteropolymer with
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205 strong auto-fluorescence in the visible as well as far-IR regions[21]. The lignin distribution in corn
206 stalks was investigated under 405 nm UV-light.
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207 Statistical error analyses: Bioethanol was produced through pretreatment, enzymolysis and
208 fermentation. Many parameters were referred consequent on this. To ensure the accuracy of data for
209 every parameters, two samples were taken out during the enzymolysis and the averages of them were
210 considered as the final sugars yield. Besides, the enzymolysis parameters as a transitional process is
211 the most important to certify the efficiency of pretreatment and provided sugars for downstream. Two
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212 parallel experiments were done. From error bar analysis, the big difference appeared at the beginning
213 and the sugar yield was similar at the end.
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214 3 Results and discussion

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215 3.1 Screening of BILs and characteristics of pretreated corn stalk

216 Since the nutrients of living organisms are biocompatible, the cations (such as choline and
217 ethanolamine) and the anions (such as glycine and acetic acid) were selected to synthesize three bionic
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218 liquids (BILs). The hydrogen bond offset from 1H NMR and FT-IR spectrua (Figure S1) indicated
219 that ethanolamine acetate ([EOA][OAc], choline acetate ([Ch][OAc], and choline glycine ([Ch][Gly])
220 were successfully synthesized. Since the three BILs have been known to be biocompatible [22], they
221 have been processed into saccharification, and the sugar yield order is found to be [Ch][Gly] >
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222 [EOA][OAc] > [Ch][OAc] (Fig.1a and b). To further explain the results, the structural changes of the
223 corn stalk were tracked before and after pretreatment.

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224 Three similar peaks are observed in the X-ray diffraction (XRD) patterns for all the samples (Fig.1c)

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225 including the main peak position at 22.7° is indicative of the distance between hydrogen-bonded
226 sheets in cellulose I [23], the broad peak at ∼16.2° is known to be a composite of two peaks from Iβ,
227 Iα, or both [24], and the third small peak at 35° corresponds to 1/4 of the length of one cellobiose unit
228 and arises from ordering along the cellulose direction [25]. The crystallinity index (CrI) is always

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229 used to express the changes between crystalline cellulose and amorphous cellulose, hemicellulose,
230 and lignin. The degrees of CrI have been considered as one of the important factors in determining
231 the hydrolysis rates of cellulosic substrates [26]. In this study, the CrI for corn stalk samples pretreated
232 by different BILs has decreased (raw corn stalk: 68% < [EOA][OAc]: 62% < [Ch][OAc]: 56% <

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233 [Ch][Gly]: 43%). The observed decrese in cellulose CrI suggests the cellulose I has been transferred
234 into cellulose II which would be easier to enzymatic hydrolysis. When CS was pretreated with [Ch]

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235 [OAc] and [Ch] [Gly], the CrI decreased from 56% to 43%. According to this, the anions of BILs
236 play a critical role in dissolving cellulose, and the inter- and intra-molecular hydrogen bonding in
237 cellulose could be effectively disrupted by accepting hydrogen bonds from cellulose hydroxyl protons
238 [27]. CrI values decrease from 62% to 56% when compared to [Ch] [OAc] and [EOA] [OAc]. It could
239
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be found that the effect of the cations is lower than the effect of anions on dissolving cellulose due to
240 the inter-hydrogen bonds in the BILs being stronger than the intra-hydrogen bonds between BILs and
241 cellulose.
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242 TGA and DSC curves show the thermal stability of pretreated corn stalks (Fig.1d and 1e). The
243 decomposition temperatures of lignin, cellulose, and hemicellulose have been reported to be 300-
244 500°C, 300-450°C, and 220-315°C, respectively [26]. Because the evaporation of moisture and
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245 volatile content in samples would disappear below 120°C, from 150°C to 790°C was considered as
246 the major decomposition section [28]. As Fig. 1c shows, the TGA curves became smoother after
247 400°C except for the raw corn stalk. It has been noticed that there was almost no compositional change
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248 (Tab. S1). Therefore, the reason for this phenomenon is the incompact structure of lignin to make
249 corn stalk pretreated by BILs easier to be depolymerized. Furthermore, the initial decomposition
250 temperatures of raw corn stalk, [EOA] [OAc], [Ch] [OAc], and [Ch] [Gly] ionic liquids were 280°C,
251 282°C, 297°C, and 275°C, respectively. The corn stalk pretreated by [Ch][Gly] has the lowest initial
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252 decomposition temperature and finishs the decomposition first.

253 From DSC analysis, the melting point of corn stalk pretreated by [Ch][Gly] is lower than that of raw
254 corn stalk, while that pretreated by [EOA][OAc] and [Ch][OAc] is higher. The trend of endothermic
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255 enthalpy agrees with that of the melting point. The decrease in endothermic enthalpy means that the
256 energy from solid to liquid was less than before. From this point, the structure of the corn stalk was
257 easier to depolymerize. The decrease in thermal stability of cellulose corresponds to the lower
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258 crystallinity of pretreated corn stalk and the results are in agreement with the XRD. Based on the

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259 enzymatic saccharification results and characteristics of pretreated CS, [Ch][Gly] was selected for

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260 further systematic investigation for the development of the one-pot bioethanol process.

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261
262
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Fig.1 BILs screening for one-pot process in term of sugar yield and structure. a) and b) performances on sugar
263 yields. Conditions: pretreatment: 140℃, 2 h, 5 wt% CS loading, 3 wt% BILs; Saccharification: 30 mL H2O,
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264 50℃, 1 MPa CO2, 35 mg enzymes; performances on structure for CS before and after pretreated by BILs
265 analyzed by XRD (c), TGA (d), and DSC (e).

266 3.2 Effect of CO2 pressure on the pH value for the pretreatment slurry
267 The pH adjustment step was necessary for the one-pot production process due to the inconsistency
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268 between [Ch][Gly] (pH=12) and enzymes (pH=4-6) [29]. The problems with using mineral or organic
269 acids for pH adjustment is that they would destroy the structure of the ionic liquid by ion exchange
270 reactions [30]. Some studies have been conducted using a volatile and easily reversible acidification
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271 agent, such as CO2, to adjust the pH of the pretreatment slurry [31]. And Sun et al. [2018] have
272 demonstrated the recovery of [Ch][Lys] ionic liquid by using this method is feasible [18]. Besides,
273 compared with imidazolium-based ionic liquids, [Ch][Gly] could be highly effective in capturing CO2
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274 due to the amido in it (0.38 mol CO2/mol IL, 298 K, 2 MPa) [32]. In this study, the pH of different
275 [Ch][Gly] concentrations under 1MPa was decreased remarkably (Fig.2a), and the pH value was
276 lower than 6, which satisfied the condition for enzymolysis when [Ch][Gly] concentration was lower
277 than 1 wt%. Hence, the concentration of [Ch][Gly] should be reduced to below 1 wt% in the
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278 subsequent one-pot bioethanol production experiments. Besides, the effects of CO2 pressure on the
279 pH of the slurry after pretreatment were studied. When CO2 pressure is increased from 1.0 MPa to
280 1.5 MPa, the glucose yield decreases from 80% to 38% at 96 h and the xylose yield decreases from
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281 70% to 30% at 96 h. When CO2 pressure is decreased to 0.6 MPa, the glucose yield decreases from
282 80% to 5% at 96 h and the xylose yield decreases from 70% to 5% at 96 h. As mentioned,CO2 can

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283 interact with amine-containing molecules via either the carbamate or carbamic acid reaction pathways

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284 [33]. The CO2 absorption by [Ch][Gly] is expected to proceed via the bicarbonate pathway in the
285 presence of water. One CO2 molecule can bind to the amine groups present in the glycine anion,
286 forming carbonic acid and in turn lowering the pH value. The solubility of CO2 increased with
287 increasing pressure to lower the pH value [34], and the pH value decreased with increasing solubility.

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288 So, it is important to choose the right pressure to give the best pH environment for enzymolysis. In
289 this sudy, 1 MPa of CO2 gives higher sugar yields.

290 On the basis of Sun et al.’s DFT calculations about the interaction between aqueous solutions of
291 [Ch][Lys] with CO2[18], the optimized structure obtained indicates that the side chain amine becomes

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292 a protonated amine when interacting with CO2 in the presence of water [35]. To further explain this

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293 phenomenon, FT-IR and 1H NMR analysis were used to explore its structure change after absorbing
294 CO2. As the N-H bond in primary amine disappears, it might have transferred into tertiary amine and
295 then the pH value decreased.
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296
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297
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298 Fig.2 Effect of 1Mpa CO2 pressure on the pH adjustment of [Ch][Gly]/H2O system (a). And effect of CO2
299 pressure on sugar yields (b) and (c). Conditions: pretreatment, 140℃, 2 h, 10 wt% CS loading, 3 wt% [Ch][Gly];

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300 Saccharification: 25 mL H2O, 50℃, 1-1.5 MPa CO2, 40mg enzymes. FT-IR (d) and 1H NMR (e) for [Ch][Gly]/

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301 [Ch][Gly]-CO2. Schematic of reversible CO2-included pH tuning for [Ch][Gly] (f).

302 3.3 Optimization of pretreatment conditions in one-pot sugars production

303 Sugar yields for the one-pot method could be affected by pretreatment parameters (e.g., corn stalk

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304 loading, temperature, time, and IL concentration) and saccharification parameters (e.g., water amount,
305 enzyme loading, temperature, and time). The effects of these parameters on sugar production were
306 investigated to maximize efficiency (Fig. 3 and Fig. 5). The sugar yields decreased from 100% to
307 60% with biomass loading during pretreatment increasing from 3 wt% to 15 wt%. These results
308 indicated that [Ch][Gly] possessed an excellent capacity for high-load biomass pretreatment

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309 compared with 5 wt% biomass loading for [BHEM]mesy ionic liquid [36]. This method would

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310 minimize production costs and maximize the effectiveness of ionic liquids. Therefore, the 10%
311 biomass loading is selected for further study by increasing saccharification time.

312 Because of the increased retention time during pretreatment, the corn stalk (CS) would be exposed to
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313 the ionic liquid aqueous for long enough to break the structure of the corn stalk. The sugar yields at
314 0.5 h were about 50%, and the sugar yield increased with retention time. However, the sugar yield
315 decreased when the retention time increased to 2.5 h, which was caused by lignin degradation. It
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316 would produce toxic aromatic molecules to restrain enzymatic activity.

317 Temperature is one critical factor affecting pretreatment efficiency. Because the lignocellulose
318 structure breaks at high temperatures, corn stalk solubility increases as the temperature rises from
319 100°C to 140°C [37]. The hydrogen bond and covalent bond between the three components of
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320 lignocellulose were destroyed during the pretreatment process, resulting in an increase in the
321 solubility of corn stalk [38]. When the temperature is increased from 140°C to 160°C, the sugar yield
322 has a plateau of about 85% glucose yield and 80% xylose yield (Fig.3c). The high temperature (more
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323 than 160°C) would change the structure of cellulose. Considering high temperature would cause the
324 high energy consumption, 140°C was selected as the optimal temperature to pretreat biomass.

325 The sugar yield of 10 wt% [Ch][Gly] was about 5% and the high [Ch][Gly] inhibited enzymatic
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326 activity significantly. When 3 wt% [Ch] [Gly] was compared to 0 wt% [Ch] [Gly], the glucose yield
327 increased from 32% to 92% and the xylose yield increased from 25% to 80%. Therefore, the
328 pretreatment procedure is important to make corn stalks fermentable into fermentable sugars. Because
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329 1 MPa of CO2 is insufficient to achieve the optimal pH for enzyme activity (Fig.2a), the sugar yield
330 gradually decreases as [Ch] [Gly] concentration increases. Imidazolium-based ILs such as
331 [C4C1Im][Cl] and [C2C1Im][OAc] are reported to deactivate cellulases when concentrations are below
332 5 wt% [20] [39, 40]. When [Ch] [Gly] concentration is 10 wt%, the glucose yield is around 40%, and
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333 the effect of [Ch] [Gly] on enzymolysis can be ignored when its pretreatment concentration is less

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334 than 3 wt%. So, the 3 wt% [Ch] [Gly] could ensure pretreatment efficiency as well as a suitable pH

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335 environment.

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336
337 Fig.3 Effect of parameters during pretreatment on one-pot sugar yields: corn stalk loading (a); time (b);
338 temperature (c); [Ch][Gly] concentration (d). Conditions: pretreatment, 140℃, 2 h, 5 wt% CS loading, 3 wt%
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339 BILs; Saccharification: 25 mL H2O, 50℃, 1 MPa CO2, 35 mg enzymes.

340 3.4 Structural changes on corn stalk pretreated by [Ch][Gly]

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341 In research about pretreatments which removed only lignin, it was found that the digestion rate of the
342 hemicellulose was similar to that of cellulose [41], due to the physical nature of the cellulose
343 components and the hemicellulose. In this case, the glucose yield and xylose yield are 80% and 79%,
344 respectively, in a one-pot process without washing and separation (Fig. 4a). These results suggest that
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345 hydrolyzing hemicellulose and cellulose at the same time could reinforce each other.

346 To further understand the reasons for one-pot sugar production, the morphology of the corn stalk
347 before and after pretreatment was observed by SEM analysis and LSCM analysis. It can be depicted
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348 from the SEM analysis (Fig. 4b) that the surface of the raw corn stalk has long and narrow cracks,
349 which indicates that stable structure of the raw corn stalk would hinder the accessibility of cellulose
350 [42]. By contrast, the surface of the pretreated corn stalk showed more cracked lines and exfoliated
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351 structure, suggesting that the CS after pretreated was broken by [Ch][Gly] and would increase the
352 accessibility between cellulose/hemicellulose and enzymes. The surface of the corn stalk after

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353 enzymolysis became asperous and smaller, which was direct evidence of the digestibility of cellulose

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354 and hemicellulose in the corn stalk. Lignin is hard to be hydrolyzed by enzymes due to its containing
355 few hydrolysable bonds, and this characteristic hinders cellulose and hemicellulose from being
356 hydrolyzed [43]. Delignification was deemed to be a method of increasing the biomass susceptibility
357 of hydrolysis. However, it has been reported that attempts to correlate the delignification of biomass

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358 to its increasing enzymolysis rate were often inconclusive or contradictory depending on variables
359 such as the types of substrate and the pretreatment conditions [44, 45]. Some papers reveal that lignin
360 distribution is more important to enzymatic hydrolysis than lignin concentration due to the complex
361 physical relationship between lignin and cellulose [46]. According to LSCM analysis (Fig.4c), the

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362 lignin distribution of raw corn stalk appears to be massive and concentrated. On the contrary, the
363 lignin distribution of pretreated corn stalks arises from tiny structures and dispersed distribution.

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364 From BET analysis, the specific surface area of the corn stalk before and after pretreatment increased
365 from 2.86 m2/g to 5.28 m2/g after pretreatment (Fig. 6e). The increase in surface area and the decrease
366 in crystallinity enhance the hydrolysis according to the relationship between the hydrolysis rate and
367 the structural features, and the most influential of the structural features is specific surface area [47].
368
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The pore diameter distribution curve was improved on the whole and the pore diameter increased
369 (Fig. 6d). The pore increase would increase accessibility between enzymes and
370 cellulose/hemicellulose. According to some studies, increasing the pore size and porosity of the
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371 subatrate may increase the likelihood of enzymolysis [48]. Therefore, increasing accessibility
372 between enzymes and cellulose/hemicellulose by increasing specific surface area, lignin distribution,
373 and pore size could enhance the hydrolysis.
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374

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375 Fig.4 The pore diameter distribution for raw and pretreated corn stalk (a). The SEM analysis for raw, pretreated

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376 and hydrolyzed corn stalk (b). The LSCM analysis for lignin distribution for raw and pretreated corn stalk (c).
377 And a comprehensive schematic diagram for corn stalk surface (d).

378 3.5 Optimization of saccharification conditions in one-pot sugars production

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379 A clear trend was observed as the sugar yields increased with time and the maximal yields were
380 obtained at 120 h (Fig. 5a and 5d). After 120 h, the sugar yields almost remained constant, and there
381 still existed some obstinate cellulose and hemicellulose that were hard to saccarfy. The amount of
382 water would affect the [Ch][Gly] concentration during the enzymolysis process, and the enzyme
383 activity of cellulase would be restrained when the [Ch][Gly] concentration is more than 1 wt% [12].

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384 The added water amount ranged from 25 mg to 45 mg to control [Ch] [Gly] concentration at 1 wt%,

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385 with 35 mg yielding the highest sugar yields. The sugar yields decreased when the water amount was
386 25 mg, 30 mg, and 45 mg. There are two reasons which may have led to this decline. One is that the
387 1 MPa CO2 is not enough to achieve the optimal pH value for enzyme action. Another is that the
388 increase in [Ch][Gly] concentration would have a suppressive effect on enzyme activity. When the
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389 water amount is 40 mg, the glucose and xylose yields are similar to those obtained when the water
390 amount is 35 mg. For the observed delicate difference with non-uniform stirring and, considering
391 saving water resources, 35 mg of water was selected for further study.
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392 When the sugar yields at 45°C and 55°C were compared, it was observed that the glucose and xylose
393 yields decreased noticeably as the temperature increased (Fig. 5b and 5e). This implies that the
394 enzyme would be sensitive to high temperatures and that there is almost no sugar released after 24 h.
395 As it is not generally agreed that the higher temperature (over 50°C) would inactivate enzymes and
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396 the lower temperature could not provide enough energy for enzymes to give full play to catalytic
397 performance, the sugar yields at 50°C are higher than others. In line with previous studies, the optimal
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398 temperature for enzymatic hydrolysis is 50°C.

399 When the enzyme loading was increased from 20 mg/g biomass to 40 mg/g biomass, glucose yield
400 and xylose yield improved from 30% to 80% and 28% to 75%, respectively (Fig. 5c and 5f). It has
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401 been known that the adsorption saturation of cellulase and substrate determines the efficiency of
402 enzymatic hydrolysis [49]. Therefore, the sugar yield increased as enzymes increased. However,
403 when the enzyme loading was increased from 40 mg/g biomass to 50 mg/g biomass, glucose yield
404 and xylose yield had no apparent difference. There are two reasons for this phenomenon: excessive
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405 enzyme cocentration and decreased enzyme activity. Besides, cellulase is essentially a multi-enzyme
406 system with polyphenism, acting on the D-glucose β-,4-glucosidic bonds in the cellulose molecules.
407 A complete cellulase system usually consists of three types of enzymes that act differently but
408 synergistically with each other, i.e., endoglucanase, exoglucanase, and β-glucosidase [50]. Therefore,
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409 40 mg of enzyme/g biomass was selected as the optimal amount.

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410
411 Fig.5 Effect of some parameters during enzymolysis on one-pot sugar yields: water amount (a and d); temperature
412 (b and e); enzymes loading (c and f). Conditions: pretreatment, 140℃, 2 h, 10 wt% CS loading, 3 wt% BILs;
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413 saccharification: 25 mL H2O, 50℃, 1 MPa CO2, 40 mg enzymes.

414 3.6 Synergy between CO2, [Ch][Gly] and enzymes for one-pot sugars production
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415 To understand the effects of CO2, [Ch][Gly] and enzymes during the one-pot process better, a series
416 of experiments including control groups were designed (Fig.6a, b, and c). The raw corn stalk hardly
417 hydrolysed with 5% sugar yields, but the corn stalks through pretreating all increased apparently. A
418 pretreatment step for subsequent effective application is necessary. Compared to enzymolysis using
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419 buffer (pH = 4.8), using CO2 to adjust pH gets the closest results. In addition, when [Ch][Gly] existed
420 in enzymatic hydrolysate, the sugar yields were higher than those without [Ch][Gly]. When carbon
421 dioxide and [Ch][Gly] existed at the same time, the enzymolysis had the best performance. Ionic
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422 liquids weaken the resistance to enzymolysis by forming stronger hydrogen bonds with the lignin of
423 corn stalks [36] (Fig.6d). Furthermore, using 1 MPa CO2 pressure in slurry after pretreatment has
424 been shown to lower pH. Cellulose and hemicellulose could continuously react with water catalyzed
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425 by enzymes (cellulase and hemicellulase). Therefore, we guessed that there is synergy between
426 carbon dioxide, [Ch][Gly] and enzymes.
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427
428 Fig.6 Comparsions of impacts of CO2 or [Ch][Gly] during enzymolysis on sugar yields (a, b and c). Conditions:
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429 pretreatment, 140℃, 2 h, 10 wt% CS loading, 3 wt% BILs; Saccharification: 25 mL H2O, 50℃, 1 MPa CO2, 40
430 mg enzymes. And Schematic of synergistic process between CO2, [Ch][Gly] and enzymes (d).

431 3.7 Universality of different biomass types for one-pot sugars production
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432 Biomass reserves (e.g. switchgrass, cotton stalks and reed stems) are plentiful in the nature, which is
433 treated as garbage instead of renewable energy sources. To explore the universality of different
434 biomass types in the one-pot method, the cotton stalk and reed stem were pretreated and hydrolyzed
435 the same way as corn stalks have been. The results showed that the glucose yields of corn stalk, cotton
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436 stalk, and reed stem were 80%, 30%, and 50% at 120 h, respectively, and the xylose yields were 80%,
437 40%, and 40%, respectively (Fig. 7a). This method is universal for different biomass types because
438 the sugar yield improves after pretreatment compared with unpretreated. However, the sugar yields
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439 were different because of different composition of each biomass (Fig. 7b). Corn stalk contains 42.6%
440 cellulose, 18.5% hemicellulose, and 14.7% lignin, respectively. Cotton stalk, on the other hand,
441 contains 29.9% cellulose, 11.5% hemicellulose, and 18.2% lignin, respectively. The contents of
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442 cellulose, hemicellulose, and lignin for reed stem are 31.4%, 18.3%, and 17.7%, respectively. The
443 higher content of lignin would make the lignocellulosic wastes hard to hydrolyze, which means the
444 higher resistance to enzymolysis in the above discussion. Otherwise, SEM analysis of different
445 biomass types (Fig. 7c) reveals that the structure of corn stalk is flaky, the structure of cotton stalk is
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446 thick, and the surface of reed stem is dense. The corn stalk would be easier to enzymolysis from
447 morphology. Based on the above analysis, the one-pot method could be applied to different types of
448 biomass, but due to the different structure and composition, corn stalk could release more sugars
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449 under the same conditions.

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450
451 Fig.7 Glucose and xylose yield for different biomass types (a and b). Conditions: pretreatment, 140℃, 2 h, 10
452 wt% CS loading, 3 wt% BILs; Saccharification: 25 mL H2O, 50℃, 1 MPa CO2, 40 mg enzymes. Composition of
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453 different biomass types (c). SEM analysis for different biomass (d).

454 3.8 Optimization of fermentation and mass balance for one-pot bioethanol production
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455 To verify the biocompatibility of [Ch][Gly] on microorganisms, the syrup after enzymolysis was
456 transferred into bioethanol by fermentation, and we researched the effects of yeast (Saccharomyces
457 cerevisiae) loading and [Ch][Gly] concentration on fermentation efficiency in a theoretical model
458 (Fig. 8a and 8b). The ethanol yield increased with yeast loading increasing, and the minimum yeast
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459 loading is 30 wt%, which could transfer glucose into bioethanol completely, and the inhibition on
460 microbial growth could be ignored when [Ch][Gly] concentration is <1 wt%. At the same
461 fermentation conditions (Tab.1), the ethanol yield could be up to 84% lower than the theoretical
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462 model. This phenomenon indicates the existence of inhitors other than [Ch] [Gly]. Therefore, becaus
463 the ethanol yield was higher than the glucose yield, it can be concluded that the saccharification and
464 fermentation were carried out simultaneously during fermentation. The one-pot bioethanol
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465 production process is repeatable because the ethanol yield in June is similar to that in August.

466 The mass balance of the one-pot [Ch][Gly] ethanol production process is displayed in Fig. 8c. During
467 the saccharification process, the 80% cellulose and hemicellulose were converted into glucose and
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468 xylose. The components of solids are lignin and others (inorganic minerals). During the fermentation
469 process, the glucose and xylose were fermented to 0.296 g of ethanol, and the ethanol concentration
470 was 8.5 g/L. Besides, the weight of solid residues after fermentation was 0.395 g, which is close to
471 the theoretical value. This indicated the mass for the whole process is conserved.
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472
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473 Fig.8 Effects of yeast loading (a) and [Ch][Gly] concentration (b) on bioethanol fermentation. Conditions: 10 wt%
474 glucose concentration, 37℃, 48 h, 20-40 wt% yeast loading. And the one-pot bioethanol production process from
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475 corn stalk by CO2 enabled using enzymes and yeast (c)

476 Tab. 1 The bioethanol yield under one-pot method in actual system*
Cellulose Hemicellulose Lignin Glucose Xylose Ethanol
Batch
(%) (%) (%) yield (%) yield (%) yield (%)
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1 (Mar.) 36.6 16.2 14.9 81.8 72.8 -

2 (Jun.) 42.6 18.5 14.7 71.1 61.4 79.0
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3 (Aug.) 42.6 18.5 14.7 78.7 66.0 84.0

477 *Under the optimal conditions as mentioned above (pretreatment, saccharification and fermentation)

4 Conclusions
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478

479 An one-pot method for producing bioethanol from corn stalk pretreated by bionic liquids was realized,
480 and adjusting the pH value of the liquid after pretreatment by CO2 was demonstrated. The key
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481 contributions in this work include: (1) [Ch] [Gly] was shown to be more effective in pretreating corn
482 stalk, with pretreated efficiency reaching 324.7 mg glucose/PU [Ch] [Gly] and 131.8 mg xylose.
483 Furthermore, due to the lower melting point, initial decomposition temperature, and crystallinity, the
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484 structure of corn stalk pretreated by [Ch] [Gly] became easier to hydrolyze; (2) the sugar yields of
485 corn stalk pretreated by [Ch] [Gly] were around 80% (glucose yield) and 79% (xylose yield), and the

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486 actual ethanol yield could be up to 84%, indicating that [Ch] [Gly] could be biocompatible with

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487 enzymes and and microorganism (Saccharomyces cerevisiae); (3) CO2 used to adjust pH avoided the
488 salt accumlation caused by mineral or organic acid and performed synergistic effect with [Ch][Gly]
489 to improve the hydrolysis efficiency; (4) the accessibility and lignin distribution were important in
490 determining the enzymatic digestibility, and the increase in surface area and pore size contributed to

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491 enhanced accessibility for enzymes to attack cellulose and hemicellulose. Overall, the one-pot method
492 by using bionic liquids could save a lot of money without washing and separation. The results
493 presented in this paper offer more possibilities for producing fermentable sugars, bioethanol and other
494 co-products derived from lignocellulose biomass based on the application of bionic liquids.

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495 Conflicts of interest

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496 There are no conflicts to declare.

497 Acknowledgements

498
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This work was financially funded by the National Key R&D Program of China (No.
499 2021YFE0190800), National Natural Science Foundation of China (Nos. 21878314, 22178343),
500 International Partnership Program of Chinese Academy of Sciences (No.122111KYSB20190060)
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501 and Hebei Natural Science Foundation (No. B2021103003), Key R&D projects in Hunan Province
502 (No. 2021SK2047), Strategic Priority Research Program of Chinese Academy of Science (No.
503 XDA21060300). This paper is based also upon work supported by the Egyptian Science, Technology,
504 and Innovation Funding Authority (STDF) under grant number 43140.
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505 Appendix A. Supplementary material

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